Delineation of the extra- and intracellular events that coordinate lineage establishment in the context of a pluripotent hematopoietic stem cell has been approached from a variety of experimental viewpoints. Using the approach of subtractive cloning, we have recently identified a novel gene product (EKLF: erythroid Kruppel-like factor) that is likely to represent an important regulator within the constellation of genes required for commitment and differentiation of erythropoietic cells. Preliminary studies indicate that EKLF expression is limited to bone marrow and spleen, the known sites of adult murine hematopoiesis. Analysis of myeloid and lymphoid cell lines reveals that EKLF expression is restricted to the erythroid cell and - at a very low level - to mast cells. The deduced amino acid sequence indicates that EKLF contains three zinc fingers most closely related to the Kruppel family of transcription factors. Together these studies strongly suggest that EKLF is an erythroid- specific transcription factor. This proposal addresses this notion by focusing on the following specific tests of EKLF molecular function: (I) Determining the EKLF target DNA binding site, using this information to implicate sites of EKLF interaction in the promoters of erythroid- restricted target genes, and demonstrating whether the presence of such a site(s) exerts a trans- activating (or repressive) effect upon a reporter gene in transfected cells; (2) Deciphering functionally important regions within the EKLF protein by testing a variety of EKLF derivatives mutated within the putative DNA binding or activation (repression) domains, and establishing whether protein-protein interactions between EKLF and other transcription factors/activators play a role in its function. These results will be extended by localizing the site of these interactions through use of the mutated proteins derived above. Establishing the molecular and biological function of EKLF, in conjunction with studies of red cell-restricted components from other labs, will help delineate how intracellular interactions of appropriately arrayed, cell-specific nuclear factors play a role in establishment and/or maintenance of the erythroid cell phenotype, and may provide an important foundation for future comparison to EKLF function in leukemic cells.